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The use of a screw for repairing defected bones is limited by the dilemma between stiffness, bioactivity and internal fixation ability in current products. For polymer bone screw, it is difficult to achieve the bone stiffness and osteo-induction. Polymer composites may enhance bioactivity and mechanical properties but sacrifice the shape memory properties enormously. Herein, we fabricated a programmable bone screw which is composed of shape memory polyurethane, hydroxyapatite and arginylglycylaspartic acid to resolve the above problem. This composite has significantly improved mechanical and shape-memory properties with a modulus of 250 MPa, a shape fixity ratio of ~90% and a shape recovery ratio of ~96%. Moreover, shape fixity and recovery ratios of the produced SMPC screw in the simulative biological condition were respectively ~80% and ~82%. The produced screw could quickly recover to its original shape in vitro within 20 s leading to easy internal fixation. Additionally, the composite could support mesenchymal stem cell survival, proliferation and osteogenic differentiation in vitro tests. It also promoted tissue growth and showed beneficial mechanical compatibility after implantation into a rabbit femoral intracondyle for 12 weeks with little inflammation. Such bone screw exhibited a fast-fixing, tightened fitting, enhanced supporting and boosted bioactivity simultaneously in the defective bone, which provides a solution to the long-standing problem for bone repairing. We envision that our composite material will provide valuable insights into the development of a new generation of bone screws with good fixation and osteogenic properties. Statement of Significance The main obstacles to a wider use of a bone screw are unsatisfied stiffness, inflammatory response and screw loosening issues. Herein, we report a programmable screw with mechanically robust, bioactive and fast-fixing performances. The shape memory polymer composite takes advantage of the component in the natural bone and possesses a stable bush-like structure inside through the covalent bonding, and thus achieve significantly improved mechanical and memory properties. Based on its shape memory effect, the produced screw was proved to offer a recovery force to surroundings and promote the bone regeneration effectively. Therefore, the composite realizes our expectations on functions through structure design and paves a practical and effective way for the development of a new generation of bone screws.

Shape memory polymers (SMPs) have great potential utility in the area of minimally invasive surgery; however, insufficient mechanical properties hinder their applications for bone defect repair, particularly in high load-bearing locations. In this study, hydroxyapatite (HA)/reduced graphene oxide (rGO) nanofillers were incorporated into a shape memory polyurethane (SMPU) to enhance its mechanical properties. Then the nanocomposite was further modified using arginyl-glycyl-aspartic acid (RGD peptide) to improve its cellular adhesion toward promoting neotissue formation and integration with surrounding bone tissue. The physical and biological properties in terms of their chemical structure, surface wettability, mechanical behaviors, shape memory performance, and cell adhesion were systematically investigated. The results demonstrated that the multimodified SMPU/HA/rGO/RGD nanocomposite significantly enhanced mechanical properties (e.g., ∼200% increase in Young's modulus and300% enhancement in tensile strength compared with the unmodified SMPU), which might be attributed to the intercalated structure and metal affinity inside the nanocomposite. Adhesion of rabbit bone mesenchymal stem cells was clearly demonstrated on an RGD-immobilized SMPU nanocomposite surface. With an excellent shape memory behavior (e.g., 97.3% of shape fixity ratio and 98.2% of shape recovery ratio), we envision that our SMPU/HA/rGO/RGD nanocomposite can be implanted into a bone defect with a minimally invasive surgery.

To summarize the research progress of interfacial tissue engineering in rotator cuff repair.The recent literature at home and abroad concerning interfacial tissue engineering in rotator cuff repair was analysed and summarized.Interfacial tissue engineering is to reconstruct complex and hierarchical interfacial tissues through a variety of methods to repair or regenerate damaged joints of different tissues. Interfacial tissue engineering in rotator cuff repair mainly includes seed cells, growth factors, biomaterials, oxygen concentration, and mechanical stimulation.The best strategy for rotator cuff healing and regeneration requires not only the use of biomaterials with gradient changes, but also the combination of seed cells, growth factors, and specific culture conditions (such as oxygen concentration and mechanical stimulation). However, the clinical transformation of the relevant treatment is still a very slow process.综述肩袖损伤修复的界面组织工程研究进展。.查阅近年国内外有关肩袖损伤修复的界面组织工程相关研究文献，并进行总结分析。.界面组织工程研究是通过多种方法重建复杂的、层次分明的界面组织，修复或再生受损的不同组织连接部位。肩袖损伤界面组织工程主要包括种子细胞、生长因子、生物材料、氧浓度和力学刺激等方面。.肩袖损伤愈合和再生的最佳策略不仅需要使用具有梯度变化的生物材料，还需要种子细胞、生长因子和特定培养条件（例如氧浓度和力学刺激）的结合，然而相应治疗手段的临床转化仍然是一个非常缓慢的过程。.

Overall high-performance shape memory polymer composites (SMCs) are desired in diverse fields while a stubborn conflict between mechanical and memory properties impedes their development in applications such as bone repair for high load-bearing locations' defects and minimally invasive surgery. For achieving a SMC with coalesced high performances in both shape memory and mechanical properties simultaneously, we here report a novel “trampoline” polyurethane nanocomposite based on the modification sequence of graphene oxide in the composite fabrication process, which can tune interlayer spacing. In this work, an intercalating structure with spring-like support, trampoline model, is proposed and was carefully examined. It is found that with larger interlayer spacing, namely taller spring support, with a longer molecular chain between two-dimensional sheets, the “trampoline” structure can remarkably improve memory and mechanical properties jointly. As desired, both high mechanical and good memory properties, e.g. ∼456.72 MPa of Young's modulus and ∼100% of shape recovery ratio, were attained respectively for the SMC. A minimally invasive bone-repairing example then demonstrated an ideal support performance for bone regeneration through finite element analysis, which showed the highest stress distributed around the implant particularly at the interface, which is essential for effective bone tissue regeneration. This “trampoline” nanocomposite paves a bright way to design and prepare SMCs with advanced performances for broad applications.

Background: Poor healing of the tendon-bone interface after rotator cuff repair is one of the main causes of surgical failure. Previous studies demonstrated that demineralized cortical bone (DCB) could improve healing of the enthesis. Purpose: To evaluate the outcomes of hierarchically demineralized cortical bone (hDCB) coated with stem cell–derived extracellular matrix (hDCB-ECM) in the repair of the rotator cuff in a rabbit model. Study Design: Controlled laboratory study. Methods: Tendon-derived stem cells (TDSCs) were isolated, cultured, and identified. Then, hDCB was prepared by the graded demineralization procedure. Finally, hDCB-ECM was fabricated via 2-week cell culture and decellularization, and the morphologic features and biochemical compositions of the hDCB-ECM were evaluated. A total of 24 rabbits (48 samples) were randomly divided into 4 groups: control, DCB, hDCB, and hDCB-ECM. All rabbits underwent bilateral detachment of the infraspinatus tendon, and the tendon-bone interface was repaired with or without scaffolds. After surgery, 8 rabbits were assessed by immunofluorescence staining at 2 weeks, and the others were assessed by micro–computed tomography (CT) examination, immunohistochemical staining, histological staining, and biomechanical testing at 12 weeks. Results: TDSCs were identified to have universal stem cell characteristics including cell markers, clonogenicity, and multilineage differentiation. The hDCB-ECM contained 3 components (bone, partial DCB, and DCB coated with ECM) with a gradient of calcium and phosphorus elements, and the ECM had stromal cell-derived factor 1, biglycan, and fibromodulin. Macroscopic observations demonstrated the absence of infection and rupture around the enthesis. The results of immunofluorescence staining showed that hDCB-ECM promoted stromal cell recruitment. Results of micro-CT analysis, immunohistochemical staining, and histological staining showed that hDCB-ECM enhanced bone and fibrocartilage formation at the tendon-bone interface. Biomechanical analysis showed that the hDCB-ECM group had higher ultimate tensile stress and Young modulus than the DCB group. Conclusion: The administration of hDCB-ECM promoted healing of the tendon-bone interface. Clinical Relevance: hDCB-ECM could provide useful information for the design of scaffolds to repair the tendon-bone interface, and further studies are needed to determine its effectiveness.

The biomechanical characteristics of tendon grafts is essential for tendon reconstructive surgery due to its great role in providing a good mechanical environment for tendon healing and regeneration. In our previous studies, the decellularized tendon slices (DTSs) and decellularized bovine tendon sheets (DBTSs) scaffolds were successfully developed. However, the influence of the integrity of tendinous membrane (endotenon and epitenon) and fascicle on biomechanical characteristics of these two scaffolds was not investigated. In this study, we assessed the integrity of tendinous membrane and fascicle of the tendon derived scaffolds and its effect on the biomechanical characteristics. The results of histological staining indicated that the DBTSs had complete endotenon and epitenon, while DTSs had no epitenon at all, only part of endotenon was remained. Furthermore, the DBTSs, and DTSs with thickness of 900 μm had complete fascicles, while DTSs with thickness less than 600 μm had almost no complete fascicles. The fibrous configuration of epitenon was well-preserved in the surface of the DBTSs but the surface ultrastructure of the DTSs was aligned collagen fibers based on scanning electron microscopy examination. The results of transmission electron microscopy showed that there was no significant difference between the DBTSs and DTSs. Mechanically, the DBTSs and DTSs with thickness of 900 μm showed similar ultimate tensile strength and stiffness to native tendon segments (NTSs). The strain at break and suture retention strength of the DBTSs showed much higher than that of the DTSs (p < 0.05). Additionally, the DBTSs showed higher ultimate load than the DTSs when these scaffolds were sutured with NTSs (p < 0.05) through the modified Kessler technique based on a uniaxial tensile test. This study demonstrated that DTSs may be used as a patch for reinforcing tendon repair, while DBTSs may be used as a bridge for reconstructing tendon defects.

Purpose The purpose of this study was to evaluate the efficacy of an extracellular matrix scaffold with multilayer decellularized tendon slices (MDTSs) for reconstructing large rotator cuff tears in a rabbit model. Methods Large defects in the infraspinatus tendons were created bilaterally in 36 rabbits. The graft group underwent bridging repair of the defects with the MDTSs grafts from Achilles tendons of adult beagle dogs, and the control group underwent repair with the autologous excised tendon. Specimens underwent histologic observation, biomechanical testing, and microcomputed tomography analysis at 2, 4, and 8 weeks after surgery. Results Histologic analysis confirmed that the MDTSs graft promoted cell ingrowth and tissue integration, and fibrocartilage and Sharpey fibers formed at the enthesis at 8 weeks. Accordingly, the MDTSs graft generated a histologic appearance similar to that of the autogenous tendon graft. Mechanical testing revealed a significant increase of the regenerated tendons in ultimate load and stiffness from 4 to 8 weeks postoperatively, which was similar to autologous tendon repair. Microcomputed tomography analysis demonstrated that the MDTSs graft promoted bone formation at the tendon-bone insertion, thus improving the mechanical properties of the repair tendon. Conclusions The MDTSs graft used to bridge large rotator cuff defects in a rabbit model promoted host cell ingrowth, enhanced the remodeling of regenerated tendon, and promoted fibrocartilage formation, thus improving the biomechanical properties of the repaired tendon. This study thereby provides fundamental information for rotator cuff regeneration with the MDTSs graft. Clinical Relevance Rotator cuff regeneration using MDTSs grafts is a promising procedure for large rotator cuff tears.

Background: Achilles tendon (AT) defects frequently occur in trauma and chronic injuries. Currently, no method can satisfactorily reconstruct the AT with completely restored function. Purpose: To evaluate the postoperative outcomes of AT defect reconstruction with decellularized bovine tendon sheets (DBTSs) in a rabbit model. Study Design: Controlled laboratory study. Methods: DBTSs were prepared from bovine tendons after compression, decellularization, antigen extraction, freeze drying, and sterilization. Platelet-rich plasma (PRP) was obtained by differential centrifugation. Sixty-three rabbits were used in this study, and the AT defect model was created bilaterally. All rabbits were divided into 3 groups (n = 21). In the DBTS group and the DBTS + PRP group, 2-cm-long AT was excised and reconstructed by DBTSs or PRP-treated DBTSs. In the control group, the rabbits underwent AT transection, and stumps were sutured. After surgery, all rabbits were assessed by ultrasonography and magnetic resonance imaging and then sacrificed for histological examination and biomechanical testing at 4, 8, or 12 weeks. Results: Gross observations demonstrated the absence of immunologic incompatibility and rejection. Histological examination showed that DBTSs promoted host cell infiltration and new fibrous tissue integration as compared with the control group. In each group, there was an AT-like structure formation and aligned collagen fiber deposition at 12 weeks. Mechanical properties of the reconstructed AT were not significantly different among the 3 groups at 4, 8, and 12 weeks after surgery ( P > .05). Ultrasonography and magnetic resonance imaging results illustrated that the reconstructed AT from each group maintained remodeling, and there was no significant difference in the echogenicity scoring ( P > .05) and percentages of good and excellent ( P > .05) among the 3 groups. Conclusion: DBTSs, which retain the native tendon structure and bioactive factors, had the ability to remodel and integrate into the rabbit AT and improve the healing process. Clinical Relevance: DBTSs could serve as an effective bioscaffold to reconstruct AT defects.

Although tissue engineering has been attracted greatly for healing of critical-sized bone defects, great efforts for improvement are still being made in scaffold design. In particular, bone regeneration would be enhanced if a scaffold precisely matches the contour of bone defects, especially if it could be implanted into the human body conveniently and safely. In this study, polyurethane/hydroxyapatite-based shape memory polymer (SMP) foam was fabricated as a scaffold substrate to facilitate bone regeneration. The minimally invasive delivery and the self-fitting behavior of the SMP foam were systematically evaluated to demonstrate its feasibility in the treatment of bone defects in vivo. Results showed that the SMP foam could be conveniently implanted into bone defects with a compact shape. Subsequently, it self-matched the boundary of bone defects upon shape-recovery activation in vivo. Micro-computed tomography determined that bone ingrowth initiated at the periphery of the SMP foam with a constant decrease towards the inside. Successful vascularization and bone remodeling were also demonstrated by histological analysis. Thus, our results indicate that the SMP foam demonstrated great potential for bone regeneration.

This study conducts a comprehensive assessment of shape memory polyurethane (SMPU) composite foams with isocyanate-modified hydroxyapatite (imHA) nanoparticles in terms of their pore structures, mechanical properties, shape memory effects and biocompatibility in vitro . The results obtained in the research reveal the effectiveness of imHA nanoparticles in SMPU foams as inorganic cross-linking fillers, which contribute to the enhancement of mechanical properties and shape memory performance. Pore structures and compressive properties are simultaneously optimized when imHA content increases. The imHA enhanced SMPU foam could be adopted as a promising alternative for overcoming the disadvantages of traditional polymer scaffolds, such as insufficient mechanical properties, inadequate pore structure, low bioactivity and inconvenience in operation for bone regeneration.

To explore a rapid histological preparation method to observe morphology and composition distribution of tendon collagen fascicle and endotendinum.Taking porcine superflexor tendon of foot as an example, tendons were sliced into sections with 6 μm by frozen section technology, after which general observation of the section integrity was carried out. After fixed with 10% neutral buffered formalin and performed with HE staining, the tissue integrity and ice crystal formation were observed under microscope. Sections were then divided into 5 groups by different methods of dyeing. Group A: Priodic acid-Shiff (PAS) staining; group B: Masson staining; group C: reticular fibers staining; group D: immunohistochemical and immunofluorescent staining of type Ⅲ collagen; group E: the sections were baked at 65℃ for 10 minutes and stained with Masson. The composition distribution of tendon collagen fascicle and endotendinum in different groups were observed.From general observation, the frozen section of tendon tissue was complete and continuous. Although the tissue integrity in the tendon sections could be seen and no ice crystal was formed, the composition distribution could not be identified by HE staining. The entire tendons in groups A, B, and C were dyed, and the composition distribution of collagen fascicle and endotendinum could not be identified. The endotendinum in group D was stained weakly positive for type Ⅲ collagen alone, and the two components were differentiated dyed but the contrast was not obvious. In group E, the collagen fascicle and endotendinium were differentiated dyed and the two components in tendon tissue were clearly visible.The morphology and the composition distribution of tendon collagen fascicle and endotendinum can be characterized rapidly and accurately, using a combination of baking at 65℃ for 10 minutes and Masson staining after porcine superflexor tendons were sliced by frozen section technology.探讨一种用于观察肌腱胶原纤维束及周围腱内膜形态与组分分布的快速组织学制片方法。.以猪趾浅屈肌腱组织为例，行冰冻切片，片厚 6 μm。大体观察切片完整性，HE 染色观察组织完整性与冰晶形成情况。然后，将切片分为 5 组，A 组行过碘酸雪夫（Priodic acid-Shiff，PAS）染色，B 组行 Masson 染色，C 组行网状纤维染色，D 组行Ⅲ型胶原免疫组织化学和免疫荧光染色，E 组切片经 65℃ 烘烤 10 min 后行 Masson 染色。观察各组肌腱胶原纤维束与周围腱内膜组分分布情况。.大体观察切片连续均匀；HE 染色见组织整体结构完整清晰，无冰晶空隙形成，但无法观察到胶原纤维束与周围腱内膜组分分布情况。A、B、C 组肌腱组织整体单一染色，无法辨认胶原纤维束与周围腱内膜组分分布；D 组周围腱内膜单独弱显色，两组分能差异化染色，但对比度不明显；E 组胶原纤维束与周围腱内膜差异化染色，两组分清晰可见。.猪趾浅屈肌腱冰冻切片后，通过 65℃ 烘烤 10 min 联合 Masson 染色方法可以快速准确显示胶原纤维束与周围腱内膜组分分布形态学表征。.

Background: Tendon-to-bone healing is a complex and slow process, and the rate of poor healing remains high. In recent years, several new strategies have been developed that enhance tendon-to-bone healing by increasing the bioactivity. Fibrin clots have been widely used to improve tissue healing and tissue engineering, Hypothesis: Modified fibrin clots can improve the bioactivity of the tendon-bone interface and histological appearance. Study Design: Controlled laboratory study. Methods: A total of 27 male New Zealand White rabbits were used. Of these, 3 were used for cell isolation, and the remaining 24 rabbits were divided into 2 groups (12 per group) for an in vivo partial patellectomy study. The setting time, degradation time, and basic fibroblast growth factor (bFGF) and ceramide-activated protein phosphatase (CaPP) release kinetics of bFGF- and CaPP-loaded fibrin clots were modified appropriately for early tendon-to-bone healing. In an in vitro experiment, the bFGF- and CaPP-loaded fibrin clots were assessed for cell migration and proliferation by microscopy, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, and DAPI (4′,6-diamidino-2-phenylindole) assay. Quantitative real-time reverse transcription polymerase chain reaction and a Western blot assay were performed to test for an induction effect of the bFGF- and CaPP-loaded fibrin clots. Finally, for the in vivo experiment, the rabbits were divided into 2 treatment groups: one with bFGF- and CaPP-loaded fibrin clots and one without bFGF- and CaPP-loaded fibrin clots after partial patellectomy in patella–patellar tendon sutured sites. A histological evaluation was performed at 2, 4, and 6 weeks after surgery. Results: The sitting time and degradation time of the bFGF- and CaPP-loaded fibrin clots were set at 15 seconds and more than 2 weeks, respectively, and the porosity was minimized to achieve the highest levels of cell migration and growth. In the bFGF-CaPP group of the in vitro experiment, cell proliferation increased to a greater extent relative to the control group ( P < .05); the mRNA expression of osteopontin, alkaline phosphatase, runt-related transcription factor 2, vascular endothelial growth factor, and collagen type I was upregulated ( P < .05); and the relative protein expression of these factors was enhanced ( P < .05). In vivo, hematoxylin and eosin staining showed that the tendon-to-bone connections were more mature and more arranged when treated with bFGF- and CaPP-loaded fibrin clots than when untreated, and the histological scores were higher. Conclusion: bFGF- and CaPP-loaded fibrin clots enhanced cell migration and proliferation and the expression of related genes and proteins, which increased the bioactivity of the tendon-bone interface and resulted in the histological improvement of tendon-to-bone healing. Clinical Relevance: As fibrin clots have already been used in clinical practice, bFGF- and CaPP-loaded fibrin clots can be further used to augment healing in the early stages of tendon-to-bone healing.

Extracellular matrix (ECM) hydrogels are used as scaffolds to facilitate the repair and reconstruction of tissues. This study aimed to optimize the decellularization process of porcine skeletal muscle ECM and to formulate a matrix hydrogel scaffold. Five multi‐step methods (methods A–E) were used to generate acellular ECM from porcine skeletal muscle [rinsing in SDS, trypsin, ethylenediaminetetraacetic acid (EDTA), Triton X‐100 and/or sodium deoxycholate at 4–37°C]. The resulting ECM was evaluated using haematoxylin and eosin, 4‐6‐diamidino‐2‐phenylindole (DAPI) staining, and DNA quantification. Acellular matrix was dissolved in pepsin and gelled at 37°C. Hydrogel response to temperature was observed in vivo and in vitro. ECM components were assessed by Masson, Sirius red, and alcian blue staining, and total protein content. Acellular porcine skeletal muscle exhibited a uniform translucent white appearance. No intact nuclear residue was detected by haematoxylin and eosin staining, while DAPI staining showed a few nuclei in the matrixes produced by methods B, C, and D. Method A generated a gel that was too thin for gelation. However, the matrix obtained by rinsing in 0.2% trypsin/0.1% EDTA, 0.5% Triton X‐100, and 1% Triton X‐100/0.2% sodium deoxycholate was nuclei‐free and produced a viscous solution that formed a structurally stable white jelly‐like hydrogel. The residual DNA content of this solution was 49.37 ± 0.72 ng/mg, significantly less than in fresh skeletal muscle, and decreased to 19.22 ± 0.85 ng/mg after gelation (P < 0.05). The acellular matrix was rich in collagen and glycosaminoglycan, with a total protein concentration of 64.8 ± 6.9%. An acellular ECM hydrogel from porcine skeletal muscle was efficiently produced.

Gels derived from decellularized small intestinal submucosa (SIS) have been used to repair ischemic myocardium and deliver protein drug. However, their material properties and effects on cell behavior are not well understood, in part because of the difficulty of gelling in vitro . In this study, soluble SIS matrix, which was easily handled and could effectively gel, was successfully prepared using a modified method. Fourier transform infrared spectroscopy confirmed that the SIS gel contained not only collagen but also sulfated glycosaminoglycans (sGAGs). Interestingly, the sustained release of vascular endothelial growth factor and basic fibroblast growth factor within the SIS gel was detected, and no initial burst release was observed. The SIS gel was more capable of evoking neovascularization than collagen type I gel, as determined by tube formation experiments in human umbilical vein endothelial cells, the mouse aortic ring assay, and animal experiments. The upregulated expression of kinase insert domain receptor (KDR), Notch1, and Ang2, the key genes in angiogenesis that were evaluated in HUVECs seeded on the SIS gel, confirmed that angiogenesis bioactive factors contained in the SIS gel are indeed active and effective. The SIS gel significantly promoted neovascularization compared to the collagen type I gel in vivo . Histology revealed adequate host tissue response in engraftment both types of gels. Together, these data demonstrate that the SIS gel is a promising and attractive candidate for tissue engineering, especially in promoting vessel formation. Statement of significance The material properties of small intestinal submucosa (SIS) gel and the effect of these properties upon cell behavior are not well understood, in part due to the difficulty of gelling in vitro . In this study, soluble SIS matrix, which was easily handled and gelled was prepared using modified method. The material properties and biocompatibility of SIS gel were explored. The sustained release of growth factors from this gel was observed along with its degradation in vitro . The results demonstrate that the SIS gel promote angiogenesis in vitro and in vivo . The SIS gel biological properties suggest that the constituent ECM molecules released from the gel remain activity. These findings suggested that the SIS gel was a promising candidate for tissue engineering, especially in promoting vessel formation.

Small intestinal submucosa (SIS)-derived gel injected into infarcted myocardium has been shown to promote repair and regeneration after myocardial infarction (MI); however, the specific impact of SIS gel on cardiomyocytes remained unknown. The aim of this study was to characterise SIS gel function in hypoxia-reoxygenation (H/R)-induced cardiomyocyte damage and its potential mechanism. HL-1 cardiomyocytes seeded on SIS matrix-coated plates, SIS gel, and uncoated plates were subjected to H/R, cell viability, apoptosis, expression of caspase-3, Bcl-2, and Bax were investigated. SIS gel and SIS matrix as coating substrates markedly improved cell viability, preventing cell apoptosis compared with uncoated plates, with SIS gel yielding the best cytoprotective effects. SIS gel down-regulated expression of pro-inflammatory cytokines (TNF-α, CCL2, and IL-6) by inhibiting the JNK-mitogen-activated protein kinase (MAPK)/NF-κB pathways. Furthermore, SIS gel protected cardiomyocytes from apoptosis by activating protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK) pathways, and markedly up-regulated antiapoptotic Bcl-2 expression but inhibited that of proapoptotic Bax and c-caspase 3. Together, these findings show that SIS gel could decrease H/R-induced cell apoptosis through a mechanism potentially related to its ability to regulate expression of inflammatory cytokines and antiapoptosis signalling pathways to prevent cell apoptosis. Our findings thereby shed light on the mechanism related to SIS gel therapeutic efficacy for MI.

We hereby report on a case in which a huge chest wall defect generated by resection of a massive aggressive tumor (desmoplastic fibroma) was repaired with osteogenic-induced mesenchymal stem cells embedded in a bone-derived biomaterial. In this case, there were three challenges to overcome: reconstruction of the soft tissue, repair of the skeletal defect of the thoracic wall and repair of the defect in the pleural cavity. The defects of soft tissue and pleural cavity were reconstructed, respectively, with an ipsilateral abdominal flap and a diaphragm muscular flap. The huge defect in the chest wall was successfully repaired with the tissue-engineered ribs, which was confirmed by long-term follow-up with computerized tomography and histological and immunohistochemical evaluations. In view of its effectiveness and safety, tissue-engineered bones may have a broad application for the repair of large skeletal defects and bone regeneration.

To develop a naturally derived tendon tissue engineering scaffold with the preservation of the native ultrastructure, tensile strength, and biochemical composition of the tendon extracellular matrix (ECM), decellularized tendon slices (DTSs) were prepared using repetitive freeze/thaw of the intact Achilles tendons, frozen section, and nuclease treatment. The DTSs were characterized in the native ultrastructure, mechanical properties, biochemical composition, and cytocompatibility. Histological examination and DNA quantification analysis confirmed that cells were completely removed from tendon tissue by repetitive freeze/thaw in combination with nuclease treatment 12 h. The intrinsic ultrastructure of tendon tissue was well preserved based on scanning electron microscopy examination. The tensile strength of the DTSs was retained 85.62% of native tendon slice. More than 93% of proteoglycans (fibromodulin, biglycan) and growth factors (TGF-β1, IGF-1, VEGF, and CTGF) inherent in tendon ECM were preserved in the DTSs according to ELISA analysis. Furthermore, the DTSs facilitated attachment and repopulation of NIH-3T3 fibroblasts in vitro. Overall, the DTSs are sheet scaffolds with a combination of elemental mechanical strength and tendon ECM bioactive factors that may have many potential applications in tendon tissue engineering.

Porcine small intestinal submucosa (SIS) has been widely used in repairing various tissues and organs. Despite this, some SIS products have the capacity to cause variable inflammatory responses after implantation resulting in severe adverse effects due to porcine cell existence. In this study, we described a multi-step method including mechanical disassociation, degrease, enzyme digestion, detergent treatment, freeze-drying and sterilization by irradiation for preparation of SIS. The efficacy of acellularization was evaluated by histological observation and the content of porcine immunoreceptor DAP12 gene. The change of growth factors contents within SIS accompanying with decellularization was quantitatively assessed by ELISA. Inflammatory reaction of SIS implanted subcutaneously in a rat was investigated. The histological examination revealed no remaining cells after enzyme digestion. Moreover, qPCR analysis demonstrated that the content of a porcine immunoreceptor gene DAP12 DNA in final SIS product (SISv) was only 1.05% of that in SIS samples (SISi) prepared by mechanical disassociation. Degrease with methanol/chloroform dramatically reduced the contents of VEGF, b-FGF, TGF-β, and TNF-α within SISii, but further treatment could not significantly reduced the contents of growth factors. SIS implanted into rats showed that inflammatory cells was more accumulated surrounded to SISi at 1, and 2 weeks, but reduced in SISv samples. The degree of inflammatory reaction for SISv was significantly less than that of SISi.

The efficacy and safety of a material derived from human bones in the repair of critical segmental bone defects are evaluated in a rhesus monkey model. Frozen human bones were chemically and physically processed into a partially demineralized and deproteinized material in blocks. The complete tissue-engineered (TE) bone was constructed of the material preseeded with allogeneic bone marrow mesenchymal stem cells (MSCs). The material alone and the TE bone were, respectively, implanted to bridge 2.5 cm-long critical defects in right and left radii of 15 monkeys. At weeks 1, 2, 3, 6 and 12 post-implantation, the grafts were collected from three animals and assessed for the local expression of osteogenic markers, histological and roentgenographic features, and immune reactions. It was shown that defects were well repaired with both treatments whereas the bone defects in 2 additional untreated animals remained the same size after 12 weeks. In radii implanted with the TE bones, the repair processes were approximately 3 weeks faster and new bones were formed in a multipoint way. There was neither observable toxic effect nor overt immune rejection in any animals. Taken together, these observations suggest that the TE bone blocks composited of the allogeneic or xenogeneic bone-derived scaffold and allogeneic MSCs may provide an ideal method for repairing large segmental bone defects.

The extracellular matrix (ECM) microenvironment for the stem cell niches, including but not limited to the biochemical composition, matrix topography, and stiffness, is crucial to stem cell proliferation and differentiation. The purpose of this study was to explore the capacity of the decellularized tendon slices (DTSs) to induce stem cell proliferation and tenogenic differentiation. Rat adult stem cells, including tendon-derived stem cells (TDSCs) and bone marrow-derived stem cells (BMSCs), were identified to have universal stem cell characteristics. The DTSs were found to retain the native tendon ECM microenvironment cues, including the inherent surface topography, well-preserved tendon ECM biochemical composition and similar stiffness to native tendon. When the TDSCs and BMSCs were cultured on the DTSs respectively, the LIVE/DEAD assay, alamarBlue® assay, scanning electron microscopy examination and qRT-PCR analysis demonstrated that the DTSs have the capacity to support these stem cells homogeneous distribution, alignment, significant proliferation and tenogenic differentiation. Taken together, the findings of this study indicate that the DTSs can provide a naturally inductive microenvironment for the proliferation and tenogenic differentiation of TDSCs and BMSCs, supporting the use of decellularized tendon ECM as a promising and valuable approach for tendon repair/reconstruction.

The effects of micropatterned surfaces coated with type I collagen (CNI) on the proliferation and morphology of rat tail tenocytes were investigated in this study. The micropatterned polydimethylsiloxane substrates were prepared by using the technique of microcontact printing and then coated with different concentrations of CNI by the microfluidic channels technology. After being seeded on the CNI-coated micropatterned substrates, the tenocytes were tested by MTT colorimetric assay at 1-, 3-, 5-, and 7-day time intervals to evaluate the proliferation of tenocytes on the substrates. The alignment and morphology of tenocytes on the CNI-coated substrates after incubation for 1 or 24 h were observed with SEM. The results showed tenocytes proliferated well with increase of CNI concentrations and identically aligned along the grooves of the CNI-coated micropatterned substrates. This could have a potential advantage in construction of engineered tendons in vitro.

To further improve surface characteristics of bio-derived compact bone scaffolds (BDCBS), 20% surface demineralization in a controlled manner was applied to the scaffolds. The surface configuration properties and roughness of the partially demineralized BDCBS and non-demineralized BDCBS (n = 12 in each group) were investigated with SEM and atomic force microscopy (AFM) in this study. The result demonstrated that the surface configuration of partially demineralized BDCBS exhibited specific porous micro-structure when compared to the compact structure of non-demineralized BDCBS. Furthermore, the result showed that the surface roughness of the partially demineralized BDCBS was significantly higher than that of BDCBS (P

To investigate the effect of canine decellularized tendon slices (DTSs) on tendon-bone healing in repairing rotator cuff injury of rabbit.Canine DTSs were prepared by repetitive freeze/thaw 5 times combined with nuclease processing for 12 hours from the adult Beagles Achilles tendons. Histological observation and cytocompatibility evaluation for the canine DTSs were performed in vitro. Twenty-four mature male New Zealand white rabbits, weighing 2.5-3.0 kg, were randomly selected. U-shaped defect of more than 50% of normal tendon in width and 8 mm in length was made in infraspinatus tendons of unilateral limb as the experimental group; the canine DTSs were used to repair defect, and the insertion of infraspinatus tendon on greater tuberosity of humerus was reconstructed in the experimental group. No treatment was done on the contralateral limb as the control group. At 4, 8, and 12 weeks after operation, the specimens were harvested for histological observation and biomechanical test.Histological examination showed that collagen fibers of canine DTSs were well preserved, without residual cells. The cytocompatibility examination showed that fibroblasts attached well to canine DTSs. Biomechanical test showed that the maximum load and stiffness increased significantly with time, and the maximum load and stiffness at 12 weeks were significantly higher than those at 4 and 8 weeks (P0.05). The maximum load and stiffness of the experimental group at 4 and 8 weeks were significantly lower than those of the control group (P0.05). The stiffness of the experimental group at 12 weeks was significantly lower than that of the control group (t = -5.679, P = 0.000), but no significant difference was found in the maximum load at 12 weeks between 2 groups (t = -0.969, P = 0.361). Histological observation showed that the control group displayed a 4-layer structure of the tendon-bone insertion. In the experimental group at 4 weeks, the tendon-bone interface was filled with granulation tissue, and a small amount of Sharpey's fibers-like connected the tendon to bone; granulation tissue disappeared, and fibroblasts, Sharpey's fiber, new cartilage, and chondrocytes significantly increased with time; tendon-bone interface became mature, but the tide line was not observed between the unmineralized fibrocartilage and mineralized fibrocartilage.Canine DTSs prepared by repetitive freeze/thaw 5 times combined with nuclease processing for 12 hours, can enhance the healing of host tendon-bone and improve the biomechanical characteristics of the rabbit infraspinatus tendon.

To investigate the effect of repeated freezing and thawing combining nuclease treatment on the decellularization of bovine tendons, and the morphology, structure, biochemical compositions, and mechanical properties of the decellularized tendons.A total of 48 fresh 1-day-old bovine Achilles tendons were randomly divided into 3 groups (n=16): fresh normal tendons (group A), repeated freezing and thawing for 5 times (liquid nitrogen refrigeration/37 degreeC thawing, group B), and repeated freezing and thawing combining nuclease processing for 24 hours (group C). In each group, 2 tendons were used for scanning electron microscope (SEM), 3 tendons for histological and immunohistochemical observations, 3 tendons for DNA content detection, and 8 tendons for biomechanical testing.SEM observation indicated the intact, aligned, and densely packed collagen fibers with no disruption in groups A and B, and the slightly loose collagen fibers with little disruption in group C. The alcian blue staining, sirius red staining, and immunohistochemical staining showed that the most of glycosaminoglycan, collagen type I, collagen type III, and fibronectin in group C were retained after decellularization treatment. HE and DAPI staining showed that the cell nuclei between the collagen fibers were clearly visible in groups A and B; however, the cell nuclei between collagen fibers almost were invisible with a few residual nuclei on the endotendineum in group C. DNA quantitative detection confirmed that DNA content in group C [(0.05 +/-0.02) micr g/mg] was significantly lower than those in group A [(0.24 +/-0.12) micro g/mg] and group B [(0.16 +/-0.07) micro g/mg] (P0.05). Biomechanical testing showed that the values of tensile strength, failure strain, stiffness, and elastic modulus were different among 3 groups, but no significant difference was found (P0.05).Repeated freezing and thawing combining nuclease processing can effectively remove the component of cells, and simultaneously retain the original collagen fibrous structure, morphology, most of the extracellular matrix compositions, and mechanical properties of the bovine tendons.

Although varieties of surgical repair techniques and materials have been used to repair rotator cuff defects, re-tearing frequently occurs. The purpose of this study is to evaluate the postoperative outcomes of rotator cuff repairs with a decellularized tendon slices (DTSs) graft in a rabbit model. Large defects in the infraspinatus tendons were created bilaterally in 21 rabbits. The graft group underwent reconstruction of the defects with the DTSs grafts, while the defect group did not undergo any treatment. The specimens underwent histological observation, biomechanical testing, and magnetic resonance imaging (MRI) detection at 4, 8, and 12 weeks after surgery. In addition, 2 rabbits that were not operated on were used for MRI detection as a normal reference. Histological analysis revealed that the graft promoted host cell ingrowth and tissue integration, and a tendon-like structure developed at 12 weeks. The ultimate tensile load had a significant difference between specimens at 4 and 12 weeks in the graft group, but there was no significant difference between the graft group and the defect group. In the graft group, the stiffness at 12 weeks was significantly greater than that at 4 or 8 weeks, and it was also greater than the stiffness in the defect group at 12 weeks. MRI demonstrated that the signal strength of the regenerative tissue from the graft group at 12 weeks was similar to that of normal infraspinatus tendon. The DTSs graft allowed for incorporation of host tendon and improved the biomechanical performance of the regenerative tendon. Therefore, the graft could be a promising bioscaffold to enhance the surgical repair of large rotator cuff defects and consequently improve the clinical outcome of rotator cuff tears.

To establish a repeatable, simple, and effective model of rat crush injury and crush syndrome.A total of 42 female Sprague Dawley rats (2-month-old, (CS) so as to lay a foundation for further study on CS. weighing 160-180 g) were divided randomly into the control group (n=6) and experimental group (n=36). The rats of the experimental group were used to establish the crush injury and CS model in both lower limbs by self-made crush injury mould. The survival rate and hematuria rate were observed after decompression. The biochemical indexes of blood were measured at 2, 4, 8, 12, 24, and 48 hours after decompression. The samples of muscle, kidney, and heart were harvested for morphological observation. There was no treatment in the control group, and the same tests were performed.Seven rats died and 15 rats had hematuria during compression in the experimental group. Swelling of the lower limb and muscle tissue was observed in the survival rats after reperfusion. The liver function test results showed that the levels of alanine transaminase and aspartate aminotransferase in the experimental group were significantly higher than those in the control group (P0.05). The renal function test results showed that blood urea nitrogen level increased significantly after 2 hours of decompression in the experimental group, showing significant difference when compared with that in the control group at 12, 24, and 48 hours after decompression (P0.05); the creatinine level of the experimental group was higher than that of the control group at 4, 8, 12, and 24 hours, showing significant difference at 8, 12, and 24 hours (P0.05). The serum K+ concentration of the experimental group was higher than that of the control group at all time, showing significant difference at the other time (P0.05) except at 2 hours. The creatine kinase level showed an increasing tendency in the experimental group, showing significant difference when compared with the level of the control group at 4, 8, 12, and 24 hours (P0.05). The histological examination of the experimental group showed that obvious edema and necrosis of the muscle were observed at different time points; glomeruli congestion and swelling, renal tubular epithelial cell degeneration, edema, necrosis, and myoglobin tube type were found in the kidneys; and myocardial structure had no obvious changes.The method of the crush injury and CS model by self-made crush injury mould is a simple and effective procedure and the experimental result is stable. It is a simple method to establish an effective model of rats crush injury and CS.

The peripheral nervous system (PNS) may be damaged by traffic accidents and natural disasters. Nerve repair and regeneration are unique clinical challenges for surgeons. A smart nerve conduit (SNC) is designed that can significantly simplify the surgery process and achieve optimal peripheral nerve regeneration (PNR) by automatic gradual lengthening. For this purpose, five macromers with different rac-lactide to glycolide weight ratios are synthesized and the characteristics of the synthesized networks are studied. Cyclic thermomechanical measurements indicate the robustness of molecular structure for shape-memory function. Body-water-responsive shape-memory behavior is evaluated by use of angle-recovery measurements. The shape-recovery time of the polymer is adjusted by selection of comonomer ratio and the overall gradual-recovery function of a device can be realized by a suitable combination of different copolymers. Thus a trisegment smart nerve conduit is fabricated from this polymer system by electrospinning and is shown to gradually recover in an in vitro experiment under stimulated physiological conditions, that is, body-liquid environment (36 °C water). In vitro culture and qualitative immunocytochemistry of Schwann cells are used to assess the biocompatibility of the fabricated SNC.

Extracellular matrix is one of the focus researches of the adipose tissue engineering. To investigate the appropriate method to prepare the porcine skeletal muscle acellular matrix and to evaluate the biocompatibility of the matrix.The fresh skeletal muscle tissues were harvested from healthy adult porcine and were sliced into 2-3 mm thick sheets, which were treated by hypotonic-detergent method to remove the cells from the tissue. The matrix was then examined by histology, immunohistochemistry, and scanning electron microscopy. The toxic effects of the matrix were tested by MTT. Human adipose-derived stem cells (hADSCs) were isolated from adipose tissue donated by patients with breast cancer, and identified by morphology, flow cytometry, and differentiation ability. Then, hADSCs of passage 3 were seeded into the skeletal muscle acellular matrix, and cultured in the medium. The cellular behavior was assessed by calcein-AM (CA) and propidium iodide (PI) staining at 1st, 3rd, 5th, and 7th days after culturing.Histology, immunohistochemistry, and scanning electron microscopy showed that the muscle fibers were removed completely with the basement membrane structure; a large number of collagenous matrix presented as regular network, porous-like structure. The cytotoxicity score of the matrix was grade 1, which meant that the matrix had good cytocompatibility. The CA and PI staining showed the seeded hADSCs had the potential of spread and proliferation on the matrix.Porcine skeletal muscle acellular matrix has good biocompatibility and a potential to be used as an ideal biomaterial scaffold for adipose tissue engineering.

To evaluate the tensile mechanical characteristics of decalcified cortical bone matrix with different thicknesses so as to provide an experimental basis for the scaffold of tissue engineering.Decalcified cortical bone matrix was prepared from fresh bovine tibia with rapid decalcification techniques. Its physical characteristics including colour, texture, and so on, were observed. Then the decalcified rate was calculated. Decalcified cortical bone matrices were radially cut into slices with different thicknesses along longitudinal axis and divided into 4 groups: group A (100-300 microm), group B (300-500 microm), group C (500-700 microm), and group D (700-1000 microm). Then the slice specimens of each group were characterized with tensile test and histological examination.General observation showed that decalcified cortical bone matrix with hydrogen peroxide treatment was ivory white with good elasticity and flexibility. The decalcified rate was 97.6%. The tensile strength and elastic modulus of groups B, C, and D were significantly higher than those of group A (P0.05); there was no significant difference among groups B, C, and D (P0.05). The stiffness in 4 groups increased gradually with the increasing thickness, it was significantly lower in group A than those in groups B, C, and D (P0.05), and in groups B and C than that in group D (P0.05). While there was no significant difference in ultimate strain within 4 groups (P0.05). Histologically, intact osteon was observed in every group, with an average maximum diameter of 182 microm (range, 102-325 microm).The mechanical properties of decalcified cortical bone matrix might depend on the integrity of the osteons. Slices with thickness of 300 microm or more could maintain similar mechanical properties when decalcified cortical bone matrix is used as a scaffold for tissue engineering.

We investigated the effects of gamma irradiation versus ethylene oxide (ETO) sterilization on the mechanical strength of cortical bone grafts. Tibias were collected from cadavers of mature goats. Sixty test specimens were randomized into four groups: fresh (no processing), frozen (freezing at -70 degrees C), gamma-irradiated, and ETO-sterilized specimens. Torsion, three-point bending, and compression testing were separately performed with a material testing machine. Parameters studied included maximum stress, strain, deflection, extension, load, shear modulus, and E-modulus. Compared with findings for the fresh specimens, findings were as follows for gamma-irradiated specimens: maximal shear modulus, reduced by 48%; shear stress, by 55%; deflection, by 71%; bending stress, by 51%; bending strain, by 74%; extension, by 60%; and compression strain, by 50%. However, there were no reductions in those parameters for the frozen specimens or the ETO-sterilized specimens. These findings confirm that shear, bending, and compression strength of cortical allografts are weakened by gamma irradiation at room temperature. To maintain optimum mechanical properties, ETO sterilization of allografts is better than gamma sterilization, especially for cortical bone, because it is usually used in load-bearing settings.

To review the progress in the treatment and research of massive rotator cuff tears.Recent literatures about the treatment and research of massive rotator cuff tears were reviewed.Treatment options of massive rotator cuff tears include nonoperative treatment, debridement, direct repair, tendon transfer, and repair with various substitutes, but the outcome is unpredictable. Recently, many experimental studies on the treatment of massive rotator cuff tears, such as gene therapy, cell therapy, and tissue engineering techniques, can provide clinicians with new treatment strategies.The treatment of massive rotator cuff tears pose a distinct clinical challenge for the orthopaedist, depending on the overall presentation. The effect of traditional operation method to repair massive rotator cuff tears is limited. The treatment and research of massive rotator cuff tears still need to be studied.

To study the possibility of bone marrow mesenchymal stem cells (BMSCs) differentiation into tenocytes (TCs) under strain stimulation by co-culture of BMSCs-small intestinal submucosa (SIS) composites in vitro.BMSCs were isolated by adherent culture from the bone marrow of 1-week-old SD rats. Inducing method of multiple differentiation and flow cytometry were applied to identify the cells. The stress-strain curve of SIS was measured with Instron machine. Purified BMSCs (2nd passage, 2.5 x 10(5) cells/cm2) were seeded on SIS (3 cm x 1 cm at size) and cultured for 2 days and then continued for another 5 days under strain stimulation (stretching frequency was 0.02 Hz, action time was 15 minutes/hour and 12 hours/day, strain amplitude was 5%) as experimental group, while the BMSCs-SIS composites were sustained static culture as control group. TCs were isolated from tail of 1-week-old SD rats. TCs-SIS composites were cultured under non-strained as positive control group. Scanning electron microscope (SEM) was used to examine the morphological changes of BMSCs after strain stimulation. The contents of Scleraxis and Tenomodulin in supernatant were tested by ELISA kit. Results The BMSCs could be induced to differentiate into osteoblasts and lipocytes, and showed the results of CD34-, CD45-, and CD90+, which were accorded with the biological characteristics of BMSCs. The failure test of SIS showed that the average elastic strain was 39.5%. SEM observation showed that the strain-stimulated BMSCs had the TCs-like morphological characteristics. The contents of Scleraxis and Tenomodulin in supernatant of experimental group, control group, and positive control group were (3.56 +/- 0.91) micromol/L and (4.27 +/- 1.10) micromol/L, (0.23 +/- 0.14) micromol/L and (0.16 +/- 0.10) micromol/L, and (14.73 +/- 2.30) micromol/L and (10.65 +/- 1.51) micromol/L, respectively. There were significant differences among 3 groups (P0.05).Appropriate strain stimulation could induce BMSCs differentiate into TCs, and the best conditions of strain stimulation need more experiments.

To review the research and application of functional tissue engineered tendons (FTETs).Recent literature concerning the research of FTETs was reviewed and analyzed.Functional tissue engineering (FTE) was a new approach that placed an emphasis on the importance of mechanical stress in determining the success of tissue engineered constructs and the effect of tissue remodeling in vivo. The concept of FTE was introduced into the research of tissue engineered tendons; by measuring in vivo loads of normal tendon and using the information as a guideline, the appropriate tissue engineered tendon which can withstand in vivo loads was designed. It would be possible to solve the problems that the biomechanical function of the tissue engineered tendons could not meet the requirements of the loading environment in vivo.FTETs have a more promising future for the treatment of tendon defects.

By observations of the features of ultrastructure changes in the tissue engineered artificial tendon of vitreous cryopreservation, we investigated the repairing effect of tendon after an in-vivo implantation and hence we provided an important theoretical and experimental basis for the vitreous cryopreservation and application of tissue engineered artificial tendon. After vitreous cryopreservation, the implantation materials of tissue engineered artificial tendon dynamically constructed in vitro were implanted in rats for reparation of the tendon defect. A scanning electron microscope was used. At the 2nd week, the materials presented a reticular formation and there were juvenile tendon cells among materials. At the 6th week, materials were already degraded and absorbed, and then were substituted by neonatal tendon cells and collagen fibers. At the 8th week, dense tendon tissues containing uniform tendon cells and collagen fibers were found already formed; the density of collagen fibers significantly increased with time. Using a transmission electron microscope at the 2nd week, we found active proliferation of tendon cells; most of them were immature cells with a complete nuclear membrane, clear nucleolus and little collagen fibers. At the 6th week, tendon cells were more mature with a little-sized, deep-stained nucleolus surrounded by plenty of collagen fibers with complete fiber structure and clear cross striation. There was no significant difference between the two groups. Using an electron microscope, we found a very good agreement in observation of the tissue engineered artificial tendon after the in-vivo implantation in two groups. Neonatal tendon cells and collagen fiber tissues grew well and are in a similar form and order when compared versus normal tendon tissues. This proved that vitreous cryopreservation has no significant influence on the function of tendon cells. The neonatal tissue-engineered tendon exerts good function of growth and repair.

To study the immunological rejection occurred in different period after the in vivo implantation of vitreous-cryopreservation tissue engineered tendons for the repair of tendon defect and investigate its influences on the hepatic, renal, and cardiovascular function of rats.Tenocytes obtained from tail tendon of one-week-old SD rats were cultured in vitro. The tenocytes at passage 2-4 (5 x 10(6) cells/mL) were co-cultured with 1.5 cm bio-derived tendon material to reconstruct tissue engineered tendon. The 21% DMSO was used as cryopreservation protection solution and the Eurocollins solution served as basic solution for pre-frozen solution (4 degrees C) and eluent. The cell-scaffold composites were vitreous-cryopreserved by self-designed method. Seventy-two healthy SD rats (male and/or female) weighing 210-230 g were randomly divided into three groups: group A (n = 32), group B (n = 32), and group C (n = 8). The 0.5 cm tendon defect model was established in the middle part of Achilles tendon in groups A and B. The defect in group A and B was repaired by the transplantation of tissue engineered tendon with and without vitreous-cryopreservation, respectively. At 2, 4, 6, and 8 weeks after transplantation, the general observation and the detection of hepatic function, renal function, and cardiovascular function were conducted. At 2, 4, and 6 weeks after transplantation, serum immunology test was conducted.There were no tissue necrosis, hydrops, and suppurative infection in groups A and B. The adhesion was evident in groups A and B 2 weeks after transplantation, improved gradually during 4-6 weeks, and disappeared at 8 weeks. The neonatal tissue had full integration and continuity, and the bridging region of the tendon healed and was similar to the normal tendon. For serum IgG and IgM content, there was no significant difference when group A or B was compared with group C, and between group A and group B 2, 4, and 6 weeks after transplantation (P0.05). Hepatic function: aspartate aminotransferase (AST) content of group A was less than that of group C 4 weeks after transplantation (P0.05); AST content of group B was less than that of group C 4 and 6 weeks after transplantation (P0.05); but there was no significant difference when group A or B was compared with group C in terms of other indexes 8 weeks after transplantation (P0.05). Renal function: serum albumin and creatinine in groups A and B were decreased obviously, and significant difference was evident when compared with group C (P0.05). Cardiovascular function: there was no significant difference between group A and group C in terms of blood glucose, triglyceride, and cholesterol (P0.05); there was a significant difference between group B and group C in terms of triglyceride 8 weeks after transplantation (P0.05).Repairing tendon defect with the implantation of vitreous-cryopreservation tissue engineered tendons results in no obvious immunological rejection and exerts no obvious influences on hepatic, renal, and cardiovascular function.

In search of a practical method for the cryopreservation of tissue engineered tendon (TET) by vitrification, we adopted 3 kinds of different cryoprotective agents (CPA)(21% DMSO, DP6 and VS55) in studying the freeze-stored effect of different CPA. The cellular morphology and post-thaw viability of the TET were examined by scanning electron microscopy (SEM), flow cytometry, and confocal laser microscopy (CLM). The results showed that there existed statistically significant difference in respect to the post-thaw viability between 21% DMSO and DP6, VS55; The cells specially adhered to the surface of scaffold both before or after cryopreservation by use of 21% DMSO. It was suggested that 21% DMSO as a CPA for TET cryopreservation was better than DP6 and VS55 in the current study.

To investigate an optimal method for SD rat skeletal muscle decellularization.Sixteen SD rats (male and female) weighing 180-200 g were used. Thirty-six skeletal muscle bundles obtained from 10 rats were randomly divided into 3 groups: normal group (group A, n=4) received non-decellularization; time group (group T, n=16) and concentration group (group C, n=16) underwent decellularization using hypotonic-detergent method. Concentration of sodium dodecyl sulfate (SDS) was 1.0% for T group, which was subdivided into groups T1, T2, T3 and T4 (n=4 per subgroup) according to different processing durations (24, 48, 72 and 96 hours). Group C was treated for 48 hours and subdivided into groups C1, C2, C3 and C4 (n=4 per subgroup) according to different SDS concentrations (0.5%, 1.0%, 1.5% and 2.0%). The muscle bundles of each group underwent HE staining observation and hydroxyproline content detection in order to get the optimal decellularization condition. Seven of 14 complete skeletal muscle bundles obtained from 6 SD rats were treated with the optimal decellularization condition (experimental group), and the rest 7 muscle bundles served as normal control (control group). The muscle bundles of each group were evaluated with gross observation, Masson staining and biomechanical test.HE staining: there was no significant difference between groups T1, T2, C1, C2 and C3 and group A in terms of muscle fiber; portion of muscle fibers in group C4 were removed; muscle fibers in group T3 were fully removed with a complete basement membrane structure; muscle fibers of group T4 were fully removed, and the structure of basement membrane was partly damaged. Hydroxyproline content detection: there was no significant difference between group A and groups C1, C2, C3, T1 and T2 (P0.05); significant difference was evident between group A and groups C4, T3 and T4 (P0.05); the difference between group C4 and groups T3 and T4 was significant (P0.05); no significant difference was evident between group T3 and group T4 (P0.05). The optimal decellularization condition was 4 degrees C, 1.0% SDS and 72 hours according to the results of HE staining and hydroxyproline content detection. Gross observation: the muscle bundles of the experimental group were pallid, half-transparent and fluffier comparing with the control group. Masson staining observation: the collagen fibers of the experimental group had a good continuity, and were fluffier comparing with control group. Biomechanics test: the maximum breaking load of the experimental group and the control group was (1.38 +/- 0.35) N and (1.98 +/- 0.77) N, respectively; the maximum extension displacement of the experimental group and the control group was (3.19 +/- 3.23) mm and (3.56 +/- 2.17) mm, respectively; there were no significant differences between two groups (P0.05).Acellular matrix with intact ECM and complete removal of muscle fibers can be obtained by oscillatory treatment of rat skeletal muscle at 4 degrees C with 1% SDS for 72 hours.

To provide the scientific theoretical basis for clinical practice by comparing biomechanical characteristics of single compressed plate with intramedullary pin, locking intramedullary nail and simple arm external fixator with simple internal fixation devices.Eighteen wet humeral bone specimens of adult cadaver were made complicated fracture models of humeral shaft and divided into 3 groups according to fixation methods. Fracture was fixed by single compressed plate with intramedullary pin in plate group, by locking intramedullary nail in intramedullary nail group and by external fixator with simple internal fixation devices in external fixator group. The intensity and rigidity of complicated fracture models of humeral shaft was measured in compress test and torsion test.In compress test, the maximum load in plate group (6,162.09 +/- 521.06) N and in intramedullary nail group (6,738.32 +/- 525.89) N was significantly larger than that in external fixator group (2,753.57 +/- 185.59) N (P0.05); but there was no significant difference between plate group and intramedullary nail group (P0.05). Under 600 N physiological compress load, the rigidity was (171.69 +/- 6.49) N/mm in plate group, (333.04 +/- 36.85) N/mm in intramedullary nail group and (132.59 +/- 2.93) N/mm in external fixator group; showing no significant difference between plate group and external fixator group (P0.05), and showing significant difference between intramedullary nail group and plate, external fixator groups (P0.05). In torsion test, the maximum torque in plate group (38.24 +/- 7.08) Nm was significantly larger than those in intramedullary nail group (17.12 +/- 5.73) Nm and external fixator group (20.26 +/- 6.42) Nm (P0.05), but there was no significant difference between intramedullary nail group and external fixator group (P0.05). Under 0.80 Nm physiological torque, the rigidity was (16.36 +/- 2.07) Ncm/degree in plate group and (18.79 +/- 2.62) Ncm/degree in external fixator group, which was significantly larger than that in intramedullary nail group (11.45 +/- 0.22) Ncm/degree (P0.05); but there was no significant difference between plate group and external fixator group (P0.05).Those fracture models fixed by single compressed plate with intramedullary pin have better compress and torsion intensity, they also have better torsion rigidity but less compress rigidity. Those fracture models fixed by locking intramedullary nail have better compress intensity but less torsion intensity, they also have better compress rigidity but less torsion rigidity. Those fracture models fixed by external fixator with simple internal fixation device have less compress and torsion intensity, they also have less compress rigidity but better torsion rigidity.

It is crucial to improve the orientation and growth of cells on substrates in tissue engineering. In this study, we investigated the effects of micropatterned surfaces coated with type I collagen (CNI) on the orientation and growth of SD rat tenocytes. Using the technique of microcontact printing and microfluidic channels, we prepared micropatterned microgrooves with a 10 microm width and 4 microm depth on silicone membrane substrates. The microgrooves were coated with CNI at concentrations 0.25, 0.5, 0.75, 1.0, and 1.25 mg/ml, respectively. The rat tenocytes at 1 x 10(5)/ml were seeded onto the CNI-coated substrates and the control substrates (without CNI coating), and then cultured in a humidified 37 degrees C/5% CO2 incubator for 48 hours. Cell proliferation was measured by MTT method. After 1, 12, 24, 48 hrs of incubation, the tenocytes' alignment and morphology were observed by means of inverted phase microscope, scanning electron microscope and fluorescent microscope. The results showed there was obvious orientation of tenocytes in CNI-modified grooves, and most of the tenocytes spread along the grooves. The tenocyte orientation became more obvious with the increasing CNI concentration over a range from 0.25 to 1.25 mg/ml. This method could find important application in the construct of engineered tendons which need precise spatial organization of cells.

To observe the changes in the peripheral blood T lymphocyte subsets and the histomorphology of the transplanted tissues in the rabbits in the early stage after transplantation of the tissue engineered bone constituted by the biologically-derived scaffold and to confirm the feasibility of the biologically-derived materials as a scaffold in the bone tissue engineering.Forty-eight healthy New Zealand rabbits (weight, 2. 0-2.5 kg) with a 1-cm defect were equally and randomly divided into 4 groups: Groups A-D. The partial demineralized freeze-dried bone (PDFDB), the tissue engineered bone constructed by the osteoblasts derived from the lactant rabbit periosteum as a seeding cell, the xenogeneic cancellous bone undergoing the antigen self-digestion, partial demineralization and freeze-dried process as a scaffold, and the fresh xenogeneic allografting bone were respectively transplanted into the segmental defects of the rabbit radii in Groups A-D. To examine the effects of the 4 different materials, the flow cytometry was used to observe the changes in the T lymphocyte subsets in the rabbit peripheral blood at 1, 2, and 4 weeks after the operations and to examine the osteogenesis achieved by the 4 materials, the histological observations were also performed at 2, 4, 8, and 12 weeks after the operations.Two weeks after the tissue engineered bone transplantation in Group B, the osteoblasts and chondroblasts were found in the apertures of the scaffold, the new bone formation could be observed, the osteoclasts could be seen in the peripheral zone, and some of the netlike frameworks were destroyed and absorbed. Four weeks after the operation, the histological observation revealed that the osteocartilagionous callus turned into a woven bone. The peripheral blood T lymphocyte subsets of CD4+ and CD8+ were significantly greater in number 1-2 weeks after the operations and in Groups A and B than before the operations and in the other groups (P0. 05);4 weeks after the operations the T lymphocyte subset of CD4+ was only slightly greater in number than before the operations, but with no statistically significant difference (P0.05). In Group C, the increase of the T lymphocyte subsets of CD4+ and CD8+ was not significant after the operation (P0.05). The T lymphocyte subsets of CD4+ and CD8+ were significantly greater in number 1, 2 and 4 weeks after the operations and in Group D than before the operation and in the other groups (P0.05).The tissue engineered bone constructed by the partial demineralized freeze-dried bone as a scaffold does not cause a serious immunologic rejection in the early stage after the transplantation and does not affect its good ability to repair the bone defect. The biologically-derived bone can be used as a scaffold in the bone tissue engineering.

A method for determining collagen in tendon by reversed-phase high performance liquid chromatography (RP-HPLC) was developed. After hydrolysis with hydrochloric acid, the collagen in samples was decomposed into hydroxyproline which hydroxyproline can be derivatized with 2, 4-dinitrochlorobenzene for the determination by HPLC (reversed-phase C18 column, 0.01 mol/L NaAc-HAc (pH 6.0)-CH3CN (80: 20, v/v) as mobile phase, detection at 360 nm). The factors influencing hydrolysis, derivatization and HPLC analysis were studied and optimized. Sixty samples were analyzed with the proposed method. The linear range was from 3 microg/L to 100 mg/L and the detection limit was 3 microg/L. The relative standard deviation (RSD) of determination was 1.95%. The recoveries of spiked samples were 98.4%-110.8%. The results show that the method is sensitive, accurate and suitable for tendon determination.

This study sought to find out a good way for the cryopreservation of tendon seeding cells so as to facilitate the preparation of tissue engineering tendons as products. The related questions are how different factors affect cell survival rate at the procedure of preservation and whether cryopreservation affects seeding cells' biological characters as well as collagen secretive function. The results of experiment indicate that DMSO is a more effective cryoprotectant in cryopreservation of tissue engineered tendon seeding cells. Blood serum nourishment is very important in cell culture, preservation and treatment. The same sustenance after cryopreservation increases cell survival rate. In the process of cryopreservation, the concentration of cells is important to cell survival rate; cell survival rate will decrease when it is less than 1.0 x 10(6)/ml. In the process of cryopreservation, the cooling speed is also important to cell survival rate, slow cooling method achieves higher cell survival rate than does the rapid cooling method. Cryopreservation by use of 10%DMSO+15%FCS+75%DMEM does not affect seeding cells' collagen secretive function greatly and does not affect seeding cells' growth curve, cell cycle and chromosome mode obviously. The prescription of 10%DMSO +15%FCS+75%DMEM is suited for the cryopreservation of tendon seeding cells.

In this brief review, some key issues related to vitreous cryopreservation of living tissues (natural or engineered), including cells, embryos, tissues, organs, and engineered tissues, are outlined. The principle of vitreous cryopreservation for the biological activity and functionality is demonstrated. The procedures of cooling/ rewarming, composition and function of optimal cryoprotectants, and their effects on bioproducts are described. Vitrification could, therefore, prove to be a useful and effective method of bioproduct cryopreservation for a long period of time, particularly for organized tissues and organs. However, the toxicity of the cryoprotective agents and the devitrification occurring during the rewarming process need additional investigations. Several key areas of research on vitrification are also addressed.

To investigate the morphology and biomechanics of in vivo osteogenesis after repairing rabbit skull defects with plastic engineered bone which was prefabricated with alginate gel, osteoblasts and bone granules.Twenty-eight rabbits were divided into group A (n=16), group B (n=8) and group C (n=4). The bilateral skull defects of 1 cm in diameter were made. Left skull defects filled with alginate gel-osteoblasts-bone granules (group A1) and right skull defects filled with alginate gel-bone granules (group A2). The defects of group B was left, as blank control and group C had no defect as normal control. The morphological change and bone formation were observed by methods of gross, histology and biomechanics.In group A1, the skull defects were almost entirely repaired by hard tissue 12 weeks after operation. The alginate gel-osteoblasts-bone granule material had changed into bone tissue with few bone granules and some residuary alginate gel. The percentage of bone formation area was 40.92% +/- 19.36%. The maximum compression loading on repairing tissue of defects was 37.33 +/- 2.95 N/mm; the maximum strain was 1.05 +/- 0.20 mm; and loading/strain ratio was 35.82 +/- 6.48 N/mm. In group A2, the alginate and bone granules material partially changed into bone tissue 12 weeks after operation. The percentage of bone formation area was 18.51% +/- 6.01%. The maximum compression loading was 30.59 +/- 4.65 N; the maximum strain was 1.35 +/- 0.44 mm; and the loading/strain ratio was 24.95 +/- 12.40 N/mm. In group B, the skull defects were mainly repaired by membrane-like soft tissue with only few bone in marginal area; the percentage of bone formation area was 12.72% +/- 9.46%. The maximum compression loading was 29.5 +/- 2.05 N; the maximum strain was 1.57 +/- 0.31 mm; and the loading/strain ratio was 19.90 +/- 5.47 N/mm. In group C, the maximum compression loading was 41.55 +/- 2.52 N; the maximum strain was 0.95 +/- 0.17 mm; and the loading/strain ratio was 47.57 +/- 11.22 N/mm.The plastic engineered bone prefabricated with alginate gel-osteoblasts-bone granule may shape according to the bone defects and has good ability to form bone tissue, whose maximum compression loading can reach 89% of normal skull and the hardness at 12 weeks after operation is similar to that of normal skull.

To investigate the feasibility of repairing goat tibia defect with marrow stromal cells (MSCs).MSCs were cocultured with the bio-derived bone in vitro, and the 20 mm tibia defects were made and fixed with plate in 35 goats, and they were divided into the experimental group, control group and blank group. The defects on the right side were filled with tissue engineering bone as the experimental group, the defects on the left side with bio-derived bone as the control group in 33 goats, and the defect on the both sides were not filled with any materials as the blank group in 2 goats. The repair capability was assessed physically, histopathologically and biomechanically at 2, 4, 6, 8, 12, 16 and 24 weeks after operation in 3 groups.By physical, histopathological and biomechanical examinations, the bio-derived bone was partially absorbed in the experimental group and was rarely absorbed in the control group in the 4th week; the defects were partially repaired in the experimental group, and in the control group, few new bones were observed in the two ends of the implants, in which there was fibrous tissue. The effects of biomechanics had no statistically significant difference between the experimental group and the control group (P0.05) in the 8th week; the defects were perfectly repaired in the experimental group and the effects of biomechanics had statistically significant difference between two groups (P0.05) in the 12th weeks. The defects were not repaired in the 24th week in the blank group.The tissue engineering bone can efficiently repair bone defect, and its repair capability is better than that of bio-derived bone alone both in quantity and in quality of bone formation.